Prevention and treatment of cardiac arrhythmias

ABSTRACT

Compositions of the invention, including compounds that bind to a receptor for a glucagon-like peptide-1, an incretin, a glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably an agonist analog), a derivative, or a variant of any of aforementioned compounds, are used in the prevention and treatment of arrhythmias associated with cardiac ischemia, cardiac ischemia-perfusion and/or congestive heart failure. The invention relates to both the method and compositions for such treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S.Provisional Application No. 60/434,508, filed Dec. 17, 2002, and U.S.Provisional Application No. 60/434,888, filed Dec. 19, 2002, which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for preventingcardiac arrhythmias using a compound that binds to a receptor for aglucagon-like peptide-1, an incretin, a glucagon-like peptide-1 (GLP-1),an exendin, or an agonist, an analog (preferably an agonist analog), aderivative, or a variant of any of aforementioned compounds andfragments thereof.

BACKGROUND OF THE INVENTION

[0003] Cardiac arrhythmias and ischemic heart disease afflict anestimated 20 million Americans, and possibly ten times as many peopleworldwide. If left undetected and untreated, they often result in heartattacks and deaths.

[0004] An arrhythmia is an irregular heartbeat. The heart beats on itsown due to its natural pacemaker, a small cluster of specialized cellscalled the sinoatrial node (S-A node). The S-A node is located in theright atrium and produces electrical signals at regular intervals thatare sent through a pathway in the heart muscle. The S-A node signalsfollow a natural electrical pathway that helps the heart beatefficiently. An electrical impulse travels from the S-A node through theatrioventricular node (A-V node), a second cluster of cells located nearthe center of the heart. The A-V node then sends the signals out to thewalls of the ventricles.

[0005] Normally, the two ventricles contract a fraction of a secondafter they have been filled with blood from an atrial contraction. Thistiming sequence is called atrio-ventricular synchrony. Sometimes,however, something goes wrong with the heart's electrical system, andthe heartbeat becomes arrhythmic. An arrhythmia can occur when: (1) theS-A node develops an abnormal rate or rhythm; (2) the normal electricalpathway is interrupted, or (3) another part of the heart tries to takeover as the pacemaker. Though there are several types of arrhythmias,they all have the commonality of preventing the heart from pumping bloodefficiently.

[0006] Fast, abnormal heart rhythms, usually over 100 beats per minute,are called tachyarrhythmias. When the heart's electrical signals comefrom the ventricle instead of the S-A node, this causes an arrhythmiaknown as ventricular tachycardia (VT). As the heart beats faster, itpumps less blood because there is not enough time for the heart to fillwith blood between beats. If this fast heartbeat continues, the brainand body may not receive enough blood and oxygen, causing faintingspells, blackouts, temporary blind spots or dizziness. Eventually, thepatient may become unconscious and in extreme cases the heart may stop(cardiac arrest). The most common cause of arrhythmias is heart disease,particularly coronary artery disease, abnormal heart valve function, andheart failure.

[0007] VT is a frequent precursor to another type of arrhythmia,ventricular fibrillation (VF). In VF, the heart beats much faster thannormal, sometimes over 300 beats a minute. The ventricles “quiver”during VF and do not carry out coordinated contractions. Because littleblood is pumped from the heart, VF is a form of cardiac arrest and isfatal unless treated immediately.

[0008] Arrhythmias complicate all forms of cardiac disease. Ventriculartachycardia and fibrillation occur commonly in the setting of ischemicheart disease and congestive heart failure (CHF). In the setting ofmyocardial infarction, ventricular arrhythmias may develop secondarilyto ischemia or reperfusion. Reperfusion occurs subsequent to therapiesthat reestablish flow in an artery that is obstructed by a blood clot,i.e. thrombolytic agents or following an intervention, such asangioplasty, coronary bypass grafting or placement of an intracoronarystent.

[0009] A major problem in congestive heart failure is stresshyperglycemia and insulin resistance. As a result of the combination ofhigh circulating levels of free fatty acids and reduced glucose uptake,there is a shift toward fatty acid oxidation, depletion of Krebs cycleintermediates and diminished glucose oxidation. These changes ultimatelylead to reduced levels of CrP and loss of energy reserve.

[0010] Although the mechanisms underlying ventricular arrhythmias arecomplex and not fully understood, it has been established thatglycolysis plays an important role as the source of ATP to maintain theelectrochemical gradient across the cardiac cellular membrane. Potassium(K⁺), calcium (Ca²⁺), and sodium (Na⁺) gradients are all modulated byATP that arises from glycolysis. Moreover, inhibition of glycolysis isarrhythmogenic, while glucose-insulin-potassium (GIK) infusions in thesetting of ischemia are anti-arrhythmic.

[0011] Conventional treatment for arrhythmias is aimed at decreasingpacemaker activity and modifying impaired conduction. These treatmentsusually involve the use of sodium channel blockers, calcium channelblockers and/or beta blockers in an effort to decrease the automaticity,conduction, and excitability of the heart or increase the refractoryperiod of cardiac muscle. While drug treatments are often effectiveagainst arrhythmias, drugs frequently have side effects and require thepatient to remember to take them on a daily basis. Mild to moderate sideeffects associated with these drugs include drowsiness, dizziness,nausea, bradycardia, and low blood pressure, while more severe sideeffects include torsades des pointes (a form of VT) and even suddendeath. Further, these drugs can actually cause arrhythmias at increaseddosages due to their toxic effects on cardiac conduction at theselevels.

[0012] Artificial pacemakers are also frequently used in the treatmentof arrhythmias. Pacemakers are electronic devices that act in place ofthe heart's own pacemaker and are programmed to imitate the normalconduction sequence of the heart. Usually they are implanted surgicallybeneath the skin of the chest and have wires running to the heart. Thereare several disadvantages associated with the use of pacemakers,including the need to replace the units every 8-10 years and theirpotential to be interfered with by certain types of equipment, such asmagnetic resonance imaging machines (MRIs).

[0013] Therapy for arrhythmias can also include devices that deliver ashock to the heart to stop an abnormal rhythm and restore a normal one.Using an electric shock for this purpose is called cardioversion,electroversion, or defibrillation. Usually in this procedure, a largemachine that delivers a shock (a defibrillator) is used by a team ofdoctors and nurses to stop a life-threatening arrhythmia. More recently,a defibrillator about the size of a pack of cards can be implantedsurgically in the patient. These small devices, which automaticallysense life-threatening arrhythmias and deliver a shock, are used inpeople who would otherwise die when their heart suddenly stops. Becausethese defibrillators do not prevent arrhythmias, the patient usuallymust also take drugs as well.

[0014] There is, therefore, a need in the art for a safe and effectivecomposition for preventing and treating cardiac arrhythmias. It is aprimary objective of this the present invention to fulfill this need.

SUMMARY OF THE INVENTION

[0015] The invention describes compositions and methods for reducing therisk of suffering from, preventing, or treating cardiac arrhythmias.Compositions of the invention include a compound that binds to areceptor for a glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of them, as well asbiologically fragments thereof.

[0016] The present inventors have recognized that compositions of theinvention, including GLP-1 and exendins, have anti-arrhythmic effects inpatients with cardiac ischemia, cardiac ischemia-reperfusion, andcongestive heart failure. For example, GLP-1 has been found to reducecardiac injury and enhance recovery in patients with these disorders.Incretins, including GLP-1, are glucose-dependent insulinotropichormones. GLP-1 and exendin effectively enhance peripheral glucoseuptake without inducing dangerous hypoglycemia. They also stronglysuppress glucagon secretion, independent of its insulinotropic action,and thereby powerfully reduce plasma free fatty acid (FFA) levelssubstantially more than can be accomplished with insulin. High FFAlevels have been implicated as a major toxic mechanism during myocardialischemia.

[0017] Accordingly, it is a primary objective of the present inventionto provide compositions and methods for preventing and treating cardiacarrhythmias.

[0018] It is a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiasthat are effective in patients having cardiac ischemia, cardiacischemia-reperfusion, and congestive heart failure.

[0019] It is yet a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiasthat reliably reduce injury associated with reperfusion and ischemia,and enhance patient recovery.

[0020] It is a further objective of the present invention to providecompositions and methods for preventing and treating cardiac arrhythmiaswithout the side effects and disadvantages of conventional therapies.

[0021] Moreover, the compounds of the invention may be administered byany conventional means, including subcutaneously, intravenously, orally,transmucosally, intraperitoneally, or other means known in the art. Thecompositions are particularly useful in treating arrhythmias resultingfrom ischemic heart disease and congestive heart failure.

[0022] Thus, in one aspect, the invention contemplates a method forpreventing and treating arrhythmias comprising administering to anindividual an effective amount of a composition which includes acompound which binds to a receptor for glucagon-like peptide-1, anincretin, a glucagon-like peptide-1 (GLP-1), an exendin, or an agonist,an analog (preferably an agonist analog), a derivative, or a variant ofany of aforementioned compounds, and biologically active fragmentsthereof.

[0023] In one embodiment, methods of the invention include administeringcompositions of the invention at a dose from about 0.1 pmol/kg/min. upto about 10 pmol/kg/min. Other dose ranges may be from about 0.01pmol/kg to 20 nmol/kg. Further contemplated are a single or multipleinjection(s) in a dose from about 0.005 nmol/kg to 20 nmol/kg.

[0024] In another embodiment, methods of the invention include aconcurrent administration with any one or more of a glucose, apotassium, a free radical scavenger or an anti-oxidant.

[0025] In yet other embodiments of the invention, the compositions ofthe invention are administered within four hours of an ischemic eventand may be continued following the ischemic event. The composition maybe administered concurrently or as soon as possible following therapiesthat reestablish flow in an artery that has been obstructed, such asangioplasty, coronary bypass grafting, and placement of an intracoronarystent.

[0026] The compositions of the invention may be administered to treatventricular arrhythmias. The ventricular arrhythmia may be caused by acondition selected from the group consisting of cardiac ischemia,cardiac ischemia-reperfusion, and congestive heart failure.

[0027] In another general aspect, methods of the invention includemetabolic intervention with a composition that includes a compound whichbinds to a receptor for glucagon-like peptide-1, an incretin, aglucagon-like peptide-1(GLP-1), an exendin, or an agonist, an analog(preferably an agonist analog), a derivative, or a variant of any ofaforementioned compounds, and fragments thereof to prevent or treatcardiac arrhythmias, said method comprising administering to anindividual in need of such treatment an effective amount of acomposition which includes a compound which binds to a receptor forglucagon-like peptide-1, an incretin, a glucagon-like peptide-1 (GLP-1),an exendin, or an agonist, an analog (preferably an agonist analog), aderivative, or a variant of any of aforementioned compounds, andbiologically active fragments thereof.

[0028] The method and means of accomplishing each of the aboveobjectives as well as others will become apparent from the detaileddescription of the invention, which follows hereafter.

DETAILED DESCRIPTION

[0029] The present invention relates to the development of compositionsfor the prevention and treatment of cardiac arrhythmias using anincretin, a glucagon-like peptide-1 (GLP-1), an exendin, a compound thatbinds to a receptor for glucagon-like peptide-1, or an agonist, ananalog (preferably an agonist analog), a derivative, or a variant of anyof the aforementioned compounds and biologically active fragmentsthereof. One premise forming the basis of this discovery is thatcompounds of the invention, including GLP-1, are effective atmaintaining the electrochemical gradient across cardiac cellularmembranes, thereby reducing the likelihood of arrhythmias developing.

[0030] Cardiac arrhythmias can develop due to a variety of factors. Forinstance, arrhythmias may develop secondary to ischemia or reperfusion.Heart muscle is largely dependent on uninterrupted blood flow, whichguarantees delivery of oxygen and substrates to cells while washing outharmful metabolic products. Ischemia, e.g. resulting from decrease orcessation of myocardial blood flow, leads to rapid changes in myocardialmetabolism. The degree of these changes is highly dependent upon theseverity of the ischemia. For anatomical and physiological reasons,contractile myocytes in endocardium are the most vulnerable cells.Ischemia is a dynamic process. With rapid reperfusion, full recovery ofmyocardial metabolism occurs; but continuation of ischemia leads tototal tissue necrosis in a few hours. Reperfusion, although generallyconsidered beneficial, can cause tissue injury by several mechanisms,including oxidative stress, and thus affect the final recovery of thecontractibility.

[0031] Total cessation of myocardial blood flow leads to rapidperturbations in myocardial metabolism. In a few seconds, oxygendissolved in cytoplasm or bound to myoglobin is consumed, seriouslydisturbing oxidative phosphorylation and mitochondrial ATP production.Levels of high energy phosphates, mainly creatine phosphate and ATP, aredecreased, and the breakdown products of adenine nucleotides, such asinorganic phosphate and adenosine, accumulate.

[0032] Liberation of free fatty acids in lipolysis is stimulated inmyocardial ischemia by increased circulating catecholamines, but fattyacid oxidation and tricarboxylic acid cycle are inhibited. This leads tocytosolic accumulation of free fatty acid CoA-esters and inhibition ofadenine nucleotide translocase. Glycogenolysis and anaerobic glycolysisare stimulated, leading to accumulation of lactate and H+-ions andintracellular acidosis. Finally, the accumulation of proteins, lactate,and reduced form of NADH leads to inhibition of glycolysis and anaerobicenergy production through glycolysis. The energy-dependent transmembranecontrol is lost, with intracellular K⁺ and Mg²⁺ ions leaking out of thecells and extracellular Na⁺ and Ca²⁺ ions entering the cells. Theredistribution of electrolytes leads to osmotic changes and cellularedema.

[0033] It is believed that several of the electrolytic changes thatoccur during ischemia may be responsible for cardiac arrhythmias. First,as noted above, during ischemia, intracellular Na⁺ increases. Duringreperfusion, this results in depolarization and short action potentialscombined with low extracellular K⁺. Dispersion is pronounced andfavorable to arrhythmias.

[0034] Second, systolic and mitochondrial Ca²⁺ levels increase duringischemia and reperfusion. An increase in cystolic Ca²⁺ activates anumber of channels, carriers, and enzymes and modulates others, whichresults in delayed afterdepolarizations and arrhythmias.

[0035] Further, during ischemia, amphiphiles and fatty acids accumulatein the plasma membrane, the gap junction, and the intracellularmembranes of the SR and the mitochondria. Amphiphiles and fatty acidsmay interact directly with channel proteins, with the phospholipidssurrounding the channel proteins, or changing the membrane fluidity.Amphiphiles increase inward current at the resting potential withsimultaneous reduction of outward current through K⁺ channels. Fattyacids activate outward currents and stimulate the K⁺/Ca²⁺ exchanger. Thesimultaneous activation of inward and outward currents favors K⁺ lossand Ca²⁺ overload, creating conditions that generate arrhythmias.(Cameliet, 1999).

[0036] It is also believed that reperfusion injury may manifestclinically as reperfusion arrhythmias. Early reperfusion is an absoluteprerequisite for the survival of ischemic tissue. Although ultimatelynecessary for recovery, reperfusion is often considered a double-edgedsword, and can actually lead to worsening of tissue injury by variousmechanisms. As with ischemia, reperfusion is associated with Ca²⁺overload through activation of the K⁺/Ca²⁺ exchanger, thereby creatingconditions favorable to cardiac arrhythmias.

[0037] Complications associated with congestive heart failure includestress hyperglycemia and insulin resistance. As a result of thecombination of high circulating levels of free fatty acids and reducedglucose uptake, there is a shift toward fatty acid oxidation. Again,these fatty acids can activate outward currents through K⁺ channels, andstimulate the K⁺/Ca²⁺ exchanger. The simultaneous activation of inwardand outward currents favors K⁺ loss and Ca²⁺ overload, thereby creatingconditions favorable to the generation of arrhythmias.

[0038] GLP-1 and exendin are glucose-dependent insulinotropic peptidesthat effectively enhance peripheral glucose uptake without inducingdangerous hypoglycemia. Further, they strongly suppress glucagonsecretion, independent of their insulinotropic action, and therebypowerfully reduce plasma free fatty acid (FFA) levels substantially morepotently than can be accomplished with insulin (i.e., greater FFAsuppression at equivalent prevailing insulin concentrations that aresubmaximally effective).

[0039] The present inventors have now discovered that GLP-1, exendins,and other compositions of the invention can be effective in theprevention and treatment of cardiac arrhythmias. It has now been foundthat the dual capacity of GLP-1 to powerfully stimulate insulin releaseand reduce insulin resistance provides this molecule with the uniqueability to prevent and treat cardiac arrhythmias by enhancing glucoseuptake and metabolism, at the expense of reduced FAA metabolism, intocardiac muscle. In this respect, incretins, GLP-1, exendins, compoundsthat bind a GLP-1 receptor, and agonists, analogs, derivatives, andvariants thereof, as well as their active fragments can be especiallyeffective in preventing and treating arrhythmias in patients withcardiac ischemia, cardiac ischemia-reperfusion, and/or congestive heartfailure.

[0040] Treatment with GLP-1 and other compositions of the invention mayenhance glycolysis in patients and shift the balance from fatty acidtowards glucose oxidation. These effects prevent loss of potassium andcalcium overload, and reduce the risk of cardiac arrhythmias.

[0041] Compositions of the invention may also stimulate the secretion ofendogenous insulin and therefore can be used to achieve all of thebeneficial actions attributed to an insulin infusion in the metabolictreatment of arrhythmias. Although high-dose GIK infusions typicallycontain 25-33% glucose and 50-100 U insulin/L, the requirement forintroduction of hyperglycemia per se to achieve therapeutic efficacy,versus only providing a metabolic milieu for the safe administration ofhigh doses of insulin, is unclear. It is likely that adequate bloodglucose levels are required to enable substrate delivery, but this doesnot necessarily imply a need for hyperglycemia and should not detractfrom the fact that insulin exerts important effects other than glucoseuptake. Therefore, a therapeutic infusion of a composition of theinvention, including GLP-1 and exendin, may require a modest (e.g., 5%)glucose administration in order to maintain blood glucose at slightlyabove physiological levels in order to trigger insulin release. Glucoseis not required as a safety measure, since blood levels of ≦3.5 mMabrogate the insulin-stimulating activity of GLP-1 and exendin, therebycompletely protecting against the dangers of hypoglycemia.

[0042] Insulin resistance (IR) has been recognized increasingly as amajor pathogenic factor for multiple systemic diseases, and not only inindividuals having Type-2 diabetes. Although many patients with Type-2diabetes manifest insulin resistance, many individuals with IR do nothave diabetes. An important recent insight has been the realization thatIR is an independent risk factor for the development and severity ofcardiovascular diseases, including ischemia-reperfusion injury and leftventricle dysfunction. IR is strongly associated with severe heartdisease, both acutely and chronically, which leads to the enhanced andpotentially damaging use by the heart of fatty acids as a fuel source inpreference to glucose. Administration of GLP-1, exendin and othercompositions of the invention, can reverse the use of fatty acids asfuel to glucose, thereby reducing free fatty acids and preventing thedevelopment of conditions favorable to the development of cardiacarrhythmias. The administration of GLP-1, exendin and other compositionsof the invention may be especially effective in the treatment ofventricular arrhythmias.

[0043] The administration of GLP-1, exendin, and other compositions ofthe invention, should be effective in a majority of patients withoutrequiring concurrent glucose administration. However, a small proportionof subjects may require glucose to elicit an adequate insulin response.In addition, it also may be necessary to administer potassium to correctexcess shifts of potassium in the intracellular compartment when glucoseis co-administered with compositions of the invention.

[0044] In addition to the use of GLP-1, exendin and other compositionsof the invention, the methods of the invention can include use of freeradical scavengers or anti-oxidants such as glutathione, melatonin,Vitamin E, and superoxide dismutase (SOD). In such combinations,reperfusion damage risk can be lessened even further.

[0045] Compositions of the invention include a compound that binds to areceptor for a glucagon like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of the aforementionedcompounds, as well as biologically active fragments thereof. An“agonist” includes any compound that mimics at least one of the actionsof an incretin, a GLP-1, or an exendin, as described herein.

[0046] An “analog” includes any peptide whose sequence was derived fromthat of the base receptor-binding compound, incretin, GLP-1, or exendin,whether or not including insertions, substitutions, extensions, ordeletions, preferably having at least 50 or 55% amino acid sequenceidentity with the base molecule, more preferably having at least 70%,80%, 90%, or 95% amino acid sequence identity with the base molecule.Such analogs may comprise conservative or non-conservative amino acidsubstitutions (including non-natural amino acids or as well as D forms),and if it is an “agonist analog,” exhibits at least one characteristicof the base molecule, preferably having a potency better than the basemolecule, or within five orders of magnitude of the base molecule, morepreferably 4, 3, 2, or 1 order of magnitude when evaluated by art-knownmeasures.

[0047] A “derivative” includes any base molecule or analog having achemical modification within, attached or linked to, or associated withthe molecule. Such chemical modifications can include internal linkers(e.g., spacing or structure-inducing) or appended molecules, such asmolecular weight-enhancing molecules (e.g., polyethylene glycol (PEG)),or tissue targeting molecules. Examples of such molecules are known inthe art, for example, insulinotropic peptides, including GLP-1 andexendin, modified with a maleimide group are described in U.S. Pat. No.6,593,295, incorporated herein by reference.

[0048] A “variant” includes any modification to the base molecule,analog or variant not encompassed in the terms “analog” and“derivative,” as would be known to a person of ordinary skill in theart. For example, variants may include proforms or chimeras of aselected molecule. Small molecules are included in the compounds usefulin the invention to the extent that they bind to a receptor for GLP-1 orexendin. Not all of the peptide molecules described as incretins,glucagon-like peptide-1 (GLP-1), exendins, or analogs, derivatives, orvariants may bind to a receptor for GLP-1, although they are stilluseful in the invention by virtue of a pharmacology not dependent on aknown GLP-1 receptor. These molecules may still possess the desiredbiological activities described herein. Other compounds encompassedwithin the scope of the invention include those described in U.S. Pat.Nos. 6,569,832; 6,528,486; 6,514,500; 6,458,924; 6,451,987; 6,451,974;6,268,343, all herein incorporated by reference.

[0049] An example of a base molecule, as the term is used above, isGLP-1, also known as glucagon-like peptide-1 [7-36] amide (also referredto as GLP-1 [7-36]NH₂), a product of the proglucagon gene having theamino acid sequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser TyrLeu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg-NH₂(SEQ ID NO:1). It is secreted into plasma mainly from the gut andproduces a variety of biological effects related to pancreatic andgastrointestinal function.

[0050] Many functions of GLP-1[7-36]NH₂, “GLP-1,” as used herein, areknown (e.g., Orskov, et al., Diabetes, 42:658-61, 1993; D'Alessio, etal., J. Clin. Invest., 97:133-38, 1996, Williams B, et al., J ClinEndocrinol Metab 81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci38 (4): 665-73, 1993; Schjoldager B T, et al., Dig Dis Sci 34 (5):703-8, 1989; O'Halloran D J, et al., J Endocrinol 126 (1): 169-73, 1990;Wettergren A, et al., Dig Dis Sci 38 (4): 665-73, 1993). GLP-1[7-37],which has an additional glycine residue at its carboxy terminus, alsostimulates insulin secretion in humans (Orskov, et al., Diabetes,42:658-61, 1993).

[0051] Compositions of the invention include GLP-1 agonist analogs. By“agonist analog” is meant a compound that mimics at least one effect ofGLP-1 as described above. This definition of agonist analog couldinclude compounds that bind to a receptor or receptors where GLP-1causes the particular effect. Certain GLP-1 analogs with agonistactivity are described in Chen et al., U.S. Pat. No. 5,512,549, issuedApr. 30, 1996, entitled Glucagon-Like Insulinotropic Peptide Analogs,Compositions and Methods of Use. Other GLP-1 analogs with agonistactivity are described in Johnson et al., U.S. Pat. No. 5,574,008,issued Nov. 12, 1996, entitled, Biologically Active Fragments ofGlucagon-Like Insulinotropic Peptide. Still other GLP-1 analogs withagonist activity are described in Buckley et al., U.S. Pat. No.5,545,618, issued Aug. 13, 1996, entitled GLP-1 Analogs Useful forDiabetes Treatment. All three referenced U.S. patents are incorporatedherein by this reference. The present invention includes the use ofrecombinant human GLP-1 analogs and GLP-1 analogs derived from otherspecies, whether recombinant or synthetic.

[0052] In certain aspects, the GLP-1 agonist analogs used in the methodsof the present invention can be GLP-1(7-34) and GLP-1(7-35), asdisclosed in U.S. Pat. No. 5,118,666, herein incorporated by reference,as well as GLP-1(7-37) as disclosed in U.S. Pat. No. 5,120,712, hereinincorporated by reference. Also included are GLP-1 analogs having areduced tendency to aggregate such as those described in WO 01/98331;GLP-1 analogs that have N-terminal truncation, U.S. Pat. No. 5,574,008;GLP-1 analogs with attached acyl groups, U.S. Pat. No. 5,512,549; andGLP-1 analogs that are amidated, WO 02/48192; and GLP-1 analogs of U.S.patent application Ser. No. 10/276772, all of which are incorporated byreference.

[0053] Additional analogs include, GLP-1 analogs at position 8, U.S.Pat. No. 5,981,488, incorporated by reference. In brief, analogs includethose of formula (XI),R₁—X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂(SEQ ID NO:33) or a pharmacuetically accetable salt thereof, wherein:

[0054] R₁ is selected from the group consisting of His, D-histidine,desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;

[0055] X is selected from the group consisting of Met, Asp, Lys, Thr,Leu, Asn, Gln, Phe, Val, and Tyr

[0056] Y and Z are independently selected from the group consisting ofGlu, Gln, Ala, Thr, Ser, and Gly, and;

[0057] R₂ is selected from the group consisting of NH₂, and Gly-OH;provided that, if R₁ is His, X is Val, Y is Glu, and Z is Glu, then R₂is NH₂.

[0058] V8-GLP-1 1and other position 8 analogs can be found in U.S. Pat.No. 5,705,483, incorporated by reference. In brief, analogs includethose of formula (XII),R₁—X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂(SEQ ID NO: 34) wherein:

[0059] R₁ is selected from the group consisting of L-histidine,D-histidine, desamino-histidine, 2-amino-histidine,.beta.-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine,and alpha-methyl-histidine;

[0060] X is selected from the group consisting of Ala, Gly, Val, Thr,Ile, and alpha-methyl-Ala;

[0061] Y is selected from the group consisting of Glu, Gln, Ala, Thr,Ser, and Gly;

[0062] Z is selected from the group consisting of Glu, Gln, Ala, Thr,Ser, and Gly;

[0063] R₂ is selected from the group consisting of NH₂, and Gly-OH;providing that the compound has an isoelectric point in the range fromabout 6.0 to about 9.0 and further providing that when R₁ is His, X isAla, Y is Glu, and Z is Glu, R₂ must be NH₂.

[0064] In other aspects, the GLP-1 agonist analogs are variants oranalogs of GLP-1 known in the art, such as, for example,Gln⁹-GLP-1(7-37), D-Gln⁹-GLP-1(7-37), acetyl-Lys⁹-GLP-1(7-37),Thr¹⁶-Lys¹⁸-GLP-1(7-37), and Lys¹⁸-GLP-1(7-37). Derivatives of GLP-1 arealso contemplated in the present invention and include, for example,acid addition salts, carboxylate salts, lower alkyl esters, and amides(see, e.g., WO91/11457). Generally, the various forms of GLP-1 are knownto stimulate insulin secretion (insulinotropic action) and cAMPformation (see, e.g., Mojsov, S., Int. J. Peptide Protein Research,40:333-343 (1992)).

[0065] In still other aspects, the present invention contemplates GLP-1agonists of the general formula (I): (SEQ ID NO: 2)      R₁-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa₄₀-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₃                                  |                                   R₂

[0066] wherein R₁ is selected from the group consisting of4-imidazopropionyl (des-amino-histidyl), 4-imidazoacetyl, or4-imidazo-alpha, alpha dimethyl-acetyl;

[0067] R₂ is selected from the group consisting of C₆-C₁₀ unbranchedacyl, or is absent;

[0068] R₃ is selected from the group consisting of Gly-OH or NH₂; and,

[0069] Xaa₄₀ is Lys or Arg.

[0070] In one embodiment, the GLP-1 agonists are naturally-occurringGLP-1(7-37) that arise from adding various R groups via a peptide bondto the amino terminus of the peptide portion of Formula I (SEQ ID NO:2).Optionally, further compounds of the invention are made by acylating theepsilon amino group of the Lys34 residue and by making limited aminoacid substitutions at position 26 or by altering the carboxy terminus.

[0071] It should be noted that for the above formula, the nomenclaturescheme used is that which has been developed around processed forms ofGLP-1. In this scheme, the amino terminus of the known GLP-1(7-37) OHhas been assigned number 7 and the carboxy terminus number 37.Therefore, the first Ala residue of Formula I corresponds to residue 8of GLP-1(7-37)OH. Likewise Xaa₄₀in Formula I corresponds to residue 26of GLP-1(7-37)OH, and so forth.

[0072] In still other aspects, the present invention providesbiologically-active GLP-1 fragments of formula (II): (SEQ ID NO: 3)      R₄-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa₄₁-Gly-Arg-R₅

[0073] wherein R₄ is selected from the group consisting of: a) H₂ N; b)H₂ N-Ser; c) H₂ N-Val-Ser; d) H₂ N-Asp-Val-Ser; e) H₂ N-Ser-Asp-Val-Ser;(SEQ ID NO: 4) f) H₂ N-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 5) g) H₂N-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 6) h) H₂N-Thr-Phe-Thr-Ser-Asp-Val- (SEQ ID NO: 7) Ser; i) H₂N-Gly-Thr-Phe-Thr-Ser-Asp- (SEQ ID NO: 8) Val-Ser; j) H₂N-Glu-Gly-Thr-Phe-Thr-Ser- (SEQ ID NO: 9) Asp-Val-Ser; or k) H₂N-Ala-Glu-Gly-Thr-Phe-Thr- (SEQ ID NO: 10) Ser-Asp-Val-Ser;

[0074] Xaa₄₁ is selected from the group consisting of Lys or Arg; and

[0075] wherein R₅ is selected from the group consisting of NH₂, OH,Gly-NH₂, or Gly-OH.

[0076] In still other aspects, the invention provides modified forms ofthe GLP-1(7-34); (7-35); (7-36) or (7-37) human peptide or theC-terminal amidated forms thereof. The native peptides have the aminoacid sequence (SEQ ID NO: 11): 7     10        15        20        25H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F    30                37-I-A-W-L-V-K-(G)-(R)-(G)

[0077] wherein (G), (R), and (G) are present or absent depending on theindicated chain length. The modified forms contain one or morealterations of the native structure and are of improved ability fortherapeutic use. Either the modified forms have greater potency thanglucagon to potentiate insulin secretion or enhanced stability in plasmaor both.

[0078] The analogs of the invention which show enhanced insulinstimulating properties may have the foregoing sequence, or a C-terminalamide thereof, with at least one modification of SEQ ID NO:11, selectedfrom the group consisting of:

[0079] (a) substitution of a neutral amino acid, arginine, or a D formof lysine for lysine at position 26 and/or 34 and/or a neutral aminoacid, lysine, or a D form of arginine for arginine at position 36;

[0080] (b) substitution of an oxidation-resistant amino acid fortryptophan at position 31;

[0081] (c) substitution according to at least one of:

[0082] Y for V at position 16;

[0083] K for S at position 18;

[0084] D for E at position 21;

[0085] S for G at position 22;

[0086] R for Q at position 23;

[0087] R for A at position 24; and

[0088] Q for K at position 26;

[0089] (d) a substitution comprising at least one of:

[0090] an alternative small neutral amino acid for A at position 8;

[0091] an alternative acidic amino acid or neutral amino acid for E atposition 9;

[0092] an alternative neutral amino acid for G at position 10; and

[0093] an alternative acidic amino acid for D at position 15; and

[0094] (e) substitution of an alternative neutral amino acid or the D orN-acylated or alkylated form of histidine for histidine at position 7.

[0095] With respect to modifications (a), (b), (d) and (e), thesubstituted amino acids may be in the D form, as indicated by asuperscript †, e.g., C\. The amino acids substituted at position 7 canalso be in the N-acylated or N-alkylated forms.

[0096] In another aspect, the invention is directed to peptides whichshow enhanced degradation resistance in plasma as compared toGLP-1(7-37) wherein this enhanced resistance to degradation is definedas set forth below. In these analogs, any of the above-mentionedtruncated forms of GLP-1(7-34) to GLP-1(7-37) or their C-terminalamidated form is modified by

[0097] (a) substitution of a D-neutral or D-acidic amino acid for H atposition 7, or

[0098] (b) substitution of a D-amino acid for A at position 8, or

[0099] (c) both, or

[0100] (d) substitution of an N-acylated or N-alkylated form of anynaturally occurring amino acid for H at position 7.

[0101] Thus, analogs of the invention which are resistant to degradationinclude (N-acyl (1-6C) AA)⁷ GLP-1(7-37) and (N-alkyl (1-6C) AA)⁷GLP-1(7-37) wherein when AA is a lysyl residue, one or both nitrogensmay be alkylated or acylated. AA symbolizes any amino acid consistentwith retention of insulin stimulating activity.

[0102] For substitutions of D-amino acids in the 7 and 8 positions ofSEQ ID NO:11, the D residue of any acidic or neutral amino acid can beused at position 7 and of any amino acid at position 8, again consistentwith insulin stimulating activity. Either or both of position 7 and 8can be substituted by a D-amino acid; the D-amino acid at position 7 canalso be acylated or alkylated as set forth above. These modified formsare applicable not only to GLP-1(7-37) but also the shorter truncatedanalogs as set forth above.

[0103] Other modified GLP-1s, as well as exendins, useful in thepractice of the claimed invention can be found in U.S. Pat. No.6,528,486, which is incorporated by reference.

[0104] As previously stated, GLP-1 analogs, as well as exendin analogs,may be peptides containing one or more amino acid substitutions,additions, extensions, or deletions, compared with GLP-1(7-36)amide,exendin-4 or exendin-3. In one embodiment, the number of substitutions,deletions, or additions is 30 amino acids or less, 25 amino acids orless, 20 amino acids or less, 15 amino acids or less, 10 amino acids orless, 5 amino acids or less or any integer in between these amounts. Inone aspect of the invention, the substitutions include one or moreconservative substitutions. A “conservative” substitution denotes thereplacement of an amino acid residue by another, biologically activesimilar residue as is well known in the art. Examples of conservativesubstitutions include the substitution of one hydrophobic residue, suchas isoleucine, valine, leucine, or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, and the like.

[0105] It is further understood that GLP-1 analogs include the abovedescribed peptides which have been chemically derivatized or altered,for example, peptides with non-natural amino acid residues (e.g.,taurine, β- and γ-amino acid residues and D-amino acid residues),C-terminal functional group modifications, such as amides, esters, andC-terminal ketone modifications and N-terminal functional groupmodifications, such as acylated amines, Schiff bases, or cyclization, asfound, for example, in the amino acid pyroglutamic acid. Exendin analogsmay have similar modifications.

[0106] Also included in the present invention are peptide sequenceshaving greater than 50% or 55% amino acid sequence identity, andpreferably greater than 70, 80, 90, or 95% amino acid sequence identityto SEQ ID NOs:1, 12, and 14, as well as truncated sequences thereof. Asused herein, sequence identity refers to a comparison made between twomolecules using standard algorithms well known in the art. The preferredalgorithm for calculating sequence identity for the present invention isthe Smith-Waterman algorithm, for example, SEQ ID NO: 1 [i.e.,GLP-1(1-37)], SEQ ID NO:12 or 14 [exendin-3 and 4, respectively] can beused as the reference sequences to define the percentage identity ofhomology over their length. The choice of parameter values for matches,mismatches, and insertions or deletions is arbitrary, although someparameter values have been found to yield more biologically realisticresults than others. One preferred set of parameter values for theSmith-Waterman algorithm is set forth in the “maximum similaritysegments” approach, which uses values of 1 for a matched residue and −⅓for a mismatched residue (a residue being either a single nucleotide orsingle amino acid). Waterman, Bull. Math. Biol. 46; 473 (1984).Insertions and deletions (indels), x, are weighted as x_(k)=1+⅓k, wherek is the number of residues in a given insert or deletion. Id.

[0107] For instance, a sequence that is identical to the 37-amino acidresidue sequence of SEQ ID NO: 1, except for 18 amino acid substitutionsand an insertion of 3 amino acids, would have a percent identity givenby:

[(1×37 matches)−(⅓×18 mismatches)−(1+3/3 indels)]/37=78% “identity.”

[0108] This algorithm can be used with any amino acid sequence todetermine sequence homology.

[0109] Agonists of glucagon-like peptide that exhibit activity through aGLP-1 (7-36)amide receptor have been described. See EP 0708179 A2;Hjorth et al., J. Biol. Chem. 269; 30121 (1994); Siegel et al., Amer.Diabetes Assoc. 57^(th) Scientific Session, Boston (1997); Hareter etal., Amer. Diabetes Assoc. 57^(th) Scientific Session, Boston (1997);Adelhorst et al., J. Biol. Chem. 269, 6275 (1994); Deacon et al.,16^(th) International Diabetes Federation Congress Abstracts,Diabetologia Supplement (1997); Irwin et al., Proc. Natl. Acad. Sci. USA94; 7915 (1997); Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992).Göke & Byrne, Diabetic Medicine 13; 854 (1996). Recent publicationsdisclose Black Widow GLP-1and Ser² GLP-1. See Holz & Hakner, Comp.Biochem. Physiol., Part B 121; 177 (1998) and Ritzel et al., J.Endocrinol 159; 93 (1998).

[0110] GLP-1 receptors are cell-surface proteins found, for example, oninsulin-producing pancreatic β-cells; the GLP-1(7-36) receptor has beencharacterised in the art. Additional receptors at which GLP-1 andexendins act are also thought to exist, and may mediate effects by whichthe instant invention is operative. Methods of determining whether achemical or peptide binds to or activates a particular GLP-1 receptorare known to the skilled artisan. For example, U.S. Pat. Nos. 6,051,689,5,846,747, and 5,670,360 describe GLP-1 receptors, as well as methodsfor using them. The contents of the patents are incorporated byreference.

[0111] The biological activity of a GLP-1 agonist and/or analog can bedetermined by in vitro and in vivo animal models and human studies, asis well known to the skilled artisan. GLP-1 biological activity can bedetermined by standard methods, in general, by receptor binding activityscreening procedures, which involve providing appropriate cells thatexpress the GLP-1 receptor on their surface, for example, insulinomacell lines such as RINmSF cells or INS-1 cells. See Mojsov, Int. J.Peptide Protein Res. 40; 333 (1992) and EP 0708179 A2. Cells that areengineered to express a GLP-1 receptor also can be used. In addition tomeasuring specific binding of tracer to membrane using radioimmunoassaymethods, cAMP activity or glucose dependent insulin-production can alsobe measured. In one method, a polynucleotide encoding a GLP-1 receptoris employed to transfect cells so that they express the GLP-1 receptorprotein. Thus, for example, these methods may be employed for screeningfor a receptor agonist by contacting such cells with compounds to bescreened and determining whether such compounds generate a signal (i.e.,activate the receptor). Other screening techniques include the use ofcells that express the GLP-1 receptor, for example, transfected CHOcells, in a system to measure extracellular pH or ionic changes causedby receptor activation. For example, potential agonists may be contactedwith a cell that expresses the GLP-1 protein receptor and a secondmessenger response (e.g., signal transduction or ionic or pH changes),may be measured to determine whether the potential agonist is effective.

[0112] Polyclonal and monoclonal antibodies can be utilized to detect,purify, and identify GLP-1-like peptides for use in the methodsdescribed herein. Antibodies such as ABGA1178 detect intact GLP-1(1-37)or N-terminally-truncated GLP-1(7-37) or GLP-1(7-36)amide. Otherantibodies detect the end of the C-terminus of the precursor molecule, aprocedure that allows one—by subtraction—to calculate the amount ofbiologically active, truncated peptide (i.e., GLP-1(7-37)amide). Orskovet al., Diabetes 42; 658 (1993); Orskov et al., J. Clin. Invest. 1991,87; 415 (1991).

[0113] GLP-1, its agonists, analogs, derivatives, variants, andbiologically active fragments, that are peptides can be made bysolid-state chemical peptide synthesis. Such peptides can also be madeby conventional recombinant techniques using standard proceduresdescribed in, for example, Sambrook & Maniatis, Molecular Cloning, ALaboratory Manual. “Recombinant,” as used herein, means that a gene isderived from a recombinant (e.g., microbial or mammalian) expressionsystem that has been genetically modified to contain a polynucleotideencoding a GLP-1 peptide as described herein.

[0114] GLP-1, its agonists, analogs, derivatives, variants, andbiologically active fragments, that are peptides may be a naturallypurified product, or a product of synthetic chemical procedures, orproduced by recombinant techniques from prokaryotic or eukaryotic hosts(for example, by bacteria, yeast, higher plant, insect, or mammaliancells in culture or in vivo). Depending on the host employed in arecombinant production procedure, the polypeptides of the presentinvention are generally non-glycosylated, but may be glycosylated. TheGLP-1 peptides can be recovered and purified from recombinant cellcultures by methods including, but not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,and lectin chromatography. High-performance liquid chromatography (HPLC)can be employed for final purification steps.

[0115] Other compositions of the invention include exendins, which referto naturally occurring exendin peptides that are found in Gila-monster.Preferred exendins include exendin-3 (SEQ ID NO:12), which is present inthe salivary secretions of Heloderma harridum, exendin-4 (SEQ ID NO:14), which is a peptide present in the salivary secretions of Helodermasuspectum (Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng.,J., et al., J. Biol. Chem., 267:7402-05, 1992), and agonists, analogs,derivative, variants of either of them as well as biologically activefragments thereof. Exendin-4, as it occurs in the salivary secretions ofthe Gila monster, is an amidated peptide. However, it should beunderstood that the term “exendin,” “exendin-3,” and “exendin-4” referto both the amidated form of the peptide and the acid form of thepeptide. Likewise, reference to GLP-1 generally refers to the amidated7-36 molecule, but it is also intended to include non-amidatedmolecules.

[0116] “Exendin agonist” refers to compounds that mimic any effect of anexendin by binding to the receptor or receptors where a naturallyoccurring exendin exerts an effect. Exendin “agonist activity” in thiscontext means having a biological activity of an exendin, such as thosedescribed herein; but it is understood that the activity of the agonistcan be either less potent or more potent than the native exendin.

[0117] Exendin-4 is a 39-amino acid polypeptide. Certain sequences arecompared in Table 1. TABLE 1 a. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (NH₂) b.HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) c.DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) d.HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) e.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPSS f.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPS g.HSDAIFTEEYSKLLAKLALQKYLASILGSRTSPPP (NH₂) h.HSDAIFTQQYSKLLAKLALQKYLASILGSRTSPPP (NH₂)

[0118] Various experiments have compared the biologic actions ofexendin-4 and GLP-1 and demonstrated a more favorable spectrum ofproperties for exendin-4. A single subcutaneous dose of exendin-4lowered plasma glucose in db/db (diabetic) and ob/ob (diabetic obese)mice by up to 40%. In Diabetic Fatty Zucker (ZDF) rats, 5 weeks oftreatment with exendin-4 lowered HbA_(1c) (a measure of glycosylatedhemoglobin used to evaluate plasma glucose levels) by up to 41%. Insulinsensitivity was also improved by 76% following 5 weeks of treatment inobese ZDF rats. In glucose intolerant primates, dose-dependent decreasesin plasma glucose were also observed.

[0119] An insulinotropic action of exendin-4 has also been observed inrodents, improving insulin response to glucose by over 100% innon-fasted Harlan Sprague Dawley (HSD) rats, and by up to ˜10-fold innon-fasted db/db mice. Higher pretreatment plasma glucose concentrationswere associated with greater glucose-lowering effects. Thus the observedglucose lowering effect of exendin-4 appears to be glucose-dependent,and minimal if animals are already euglycemic. Degradation studies withexendin-4 compared to GLP-1 indicate that exendin-4 is relativelyresistant to degradation.

[0120] As used in this specification, the term “exendin agonist”includes any molecules, whether they be peptides, peptide mimetics, orother chemical compounds, that bind to or activate a receptor orreceptors at which exendin exerts an effect, as described above.Moreover, exendin agonists may include molecules having insulinotropicactivity and that may bind a GLP-1 receptor molecule in in vitro assaysand induce second messenger activity on, inter alia, insulin producingβ-cells.

[0121] The structure activity relationship (SAR) of exendin wasinvestigated for structures that may relate to the activity of exendin,for its stability to metabolism, and for improvement of its physicalcharacteristics, especially as it pertains to peptide stability and toamenability to alternative delivery systems, and various exendin agonistpeptide compounds have been invented. Exendin agonists include exendinanalogs with agonist activity in which one or more naturally ornon-naturally occurring amino acids are added, inserted, eliminated orreplaced with another amino acid(s). Preferred exendin analogs arepeptide analogs of exendin-4.

[0122] Exendin analogs include peptides that are encoded bypolynucleotides that express biologically active exendin analogs withagonist activity, as defined herein. For instance, exendin analogs maybe peptides containing one or more amino acid substitutions, extensions,additions or deletions, compared with exendin-4 or exendin-3. In oneembodiment, the number of substitutions, extension, deletions, oradditions is 30 amino acids or less, 25 amino acids or less, 20 aminoacids or less, 15 amino acids or less, 10 amino acids or less, 5 aminoacids or less or any integer in between these amounts. In one aspect ofthe invention, the substitutions include one or more conservativesubstitutions. Exendin analogs, which include chemically derivatized oraltered compounds and peptides having a preferred amino acid homology toSEQ ID NOs:12 and 14 have been previously described and are contemplatedto be within the scope of the claimed invention.

[0123] Novel exendin analogs with agonist activity are described in PCTApplication Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997, both ofwhich are herein incorporated by reference.

[0124] Other novel exendin analogs with agonist activity are describedin PCT Application Serial No. PCT/US98/24210, filed Nov. 13, 1998,entitled “Novel Exendin Agonist Compounds,” which claims the benefit ofU.S. Provisional Application No. 60/065,442 filed Nov. 14, 1997, both ofwhich are herein incorporated by reference.

[0125] Still other novel exendin analogs with agonist activity aredescribed in PCT Application Serial No. PCT/US98/24273, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds,” which claims thebenefit of U.S. Provisional Application No. 60/066,029 filed Nov. 14,1997, both of which are herein incorporated by reference.

[0126] Still other exendin analogs with agonist activity are describedin PCT Application Serial No. PCT/US97/14199, filed Aug. 8, 1997,entitled “Methods for Regulating Gastrointestinal Activity,” which is acontinuation-in-part of U.S. patent application Ser. No. 08/694,954filed Aug. 8, 1996, both of which are hereby incorporated by reference.

[0127] Still other exendin analogs with agonist activity are describedin PCT Application Serial No. PCT/US98/00449, filed Jan. 7, 1998,entitled “Use of Exendins and Agonists Thereof for the Reduction of FoodIntake,” which claims priority to U.S. Provisional Application No.60/034,905 filed Jan. 7, 1997, both of which are hereby incorporated byreference.

[0128] Activity as exendin agonists and exendin analogs with agonistactivity can be indicated, for example, by activity in the assaysincorporated by reference in the referenced applications. Effects ofexendins or exendin agonists can be identified, evaluated, or screenedfor, using the methods described herein, or other art-known orequivalent methods for determining the effects of exendin. Screeningassays for potential exendin agonist compounds or candidate exendinagonist compounds, may include an in vitro GLP-1 receptor assay/screendescribed above, an amylin receptor assay/screen using an amylinreceptor preparation as described in U.S. Pat. No. 5,264,372, issuedNov. 23, 1993, the contents of which are incorporated herein byreference, one or more calcitonin receptor assays/screens using, forexample, T47D and MCF7 breast carcinoma cells, which contain calciumreceptors coupled to the stimulation of adenyl cyclase activity, and/ora CGRP receptor assay/screen using, for example, SK-N-MC cells.

[0129] Certain preferred exendin analogs with agonist activity include:

[0130] exendin-4 (1-30) [SEQ ID NO:19: His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp LeuLys Asn Gly Gly];

[0131] exendin-4 (1-30) amide [SEQ ID NO:20: His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Gly-NH₂];

[0132] exendin-4 (1-28) amide [SEQ ID NO:21: His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn-NH₂];

[0133]¹⁴Leu,²⁵Phe exendin-4 amide [SEQ ID NO:22: His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gin Leu Glu Glu Glu Ala Val Arg Leu Phe Ile GluPhe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH₂];

[0134]¹⁴Leu,²⁵Phe exendin-4 (1-28) amide [SEQ ID NO:23: His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu PheIle Glu Phe Leu Lys Asn-NH₂]; and

[0135]¹⁴Leu, ²²Ala, ²⁵Phe exendin-4 (1-28) amide [SEQ ID NO:24: His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val ArgLeu Ala Ile Glu Phe Leu Lys Asn-NH₂].

[0136] Also included within the scope of the present invention arepharmaceutically acceptable salts of the compounds of formula (III-X)and pharmaceutical compositions including said compounds and saltsthereof.

[0137] Formula III

[0138] Exendin analogs with agonist activity also include thosedescribed in U.S. Provisional Application No. 60/065,442, includingcompounds of the formula (III) [SEQ ID NO:25]:

[0139] Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;

[0140] wherein

[0141] Xaa₁ is His, Arg or Tyr;

[0142] Xaa₂ is Ser, Gly, Ala or Thr;

[0143] Xaa₃ is Asp or Glu;

[0144] Xaa₅ is Ala or Thr;

[0145] Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

[0146] Xaa₇ is Thr or Ser;

[0147] Xaa₈ is Ala, Ser or Thr;

[0148] Xaa₉ is Asp or Glu;

[0149] Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

[0150] Xaa₁₁ is Ala or Ser;

[0151] Xaa₁₂ is Ala or Lys;

[0152] Xaa₁₃ is Ala or Gln;

[0153] Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

[0154] Xaa₁₅ is Ala or Glu;

[0155] Xaa₁₆ is Ala or Glu;

[0156] Xaa₁₇ is Ala or Glu;

[0157] Xaa₁₉ is Ala or Val;

[0158] Xaa₂₀ is Ala or Arg;

[0159] Xaa₂₁ is Ala or Leu;

[0160] Xaa₂₂ is Ala, Phe, Tyr or naphthylalanine;

[0161] Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0162] Xaa₂₄ is Ala, Glu or Asp;

[0163] Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

[0164] Xaa₂₆ is Ala or Leu;

[0165] Xaa₂₇ is Ala or Lys;

[0166] Xaa₂₈ is Ala or Asn;

[0167] Z₁ is —OH,

[0168] —NH₂

[0169] Gly-Z₂,

[0170] Gly Gly-Z₂,

[0171] Gly Gly Xaa₃₁-Z₂,

[0172] Gly Gly Xaa₃₁ Ser-Z₂,

[0173] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0174] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0175] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0176] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0177] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or

[0178] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

[0179] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro, homoproline,3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine; and

[0180] Z₂ is —OH or —NH₂;

[0181] provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala.

[0182] Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycineand N-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms.

[0183] Preferred exendin analogs include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

[0184] Preferred are those compounds wherein Xaa₂ is Gly.

[0185] Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycineor Met.

[0186] Preferred compounds are those wherein Xaa₂₅ is Trp or Phe.

[0187] Preferred compounds are those where Xaa₆ is Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine and

[0188] Xaa₂₃ is Ile or Val.

[0189] Preferred are compounds wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline andN-alkylalanine.

[0190] Preferably Z₁ is —NH₂.

[0191] Preferably Z₂ is —NH₂.

[0192] According to one aspect, preferred are compounds of formula (III)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ isPhe or naphthylalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Pheor naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈are independently selected from Pro, homoproline, thioproline orN-alkylalanine. More preferably Z₁ is —NH₂.

[0193] According to an especially preferred aspect, especially preferredcompounds include those of formula (III) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₅ is Ala or Thr; Xaa₆ is Ala,Phe or nephthylalaine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉is Asp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala or Ser;Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu orpentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ SerSer-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Pro homoproline,thioproline or N-methylalanine; and Z₂ being —OH or —NH₂; provided thatno more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂,Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ andXaa₂₈ are Ala. Especially preferred compounds include those set forth inPCT application Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” identified therein as compounds 2-23.

[0194] According to an especially preferred aspect, provided arecompounds where Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferablyLeu or pentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, morepreferably Phe or naphthylalanine. These compounds will be lesssusceptive to oxidative degration, both in vitro and in vivo, as well asduring synthesis of the compound.

[0195] Formula IV

[0196] Exendin analogs with agonist activity also include thosedescribed in U.S. Provisional Application No. 60/066,029, includingcompounds of the formula (IV)[SEQ ID NO:26]:

[0197] Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z¹;

[0198] wherein:

[0199] Xaa₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu;

[0200] Xaa₂ is Ser, Gly, Ala or Thr;

[0201] Xaa₃ is Ala, Asp or Glu;

[0202] Xaa₄ is Ala, Norval, Val, Norleu or Gly;

[0203] Xaa₅ is Ala or Thr;

[0204] Xaa₆ is Phe, Tyr or naphthylalanine;

[0205] Xaa₇ is Thr or Ser;

[0206] Xaa₈ is Ala, Ser or Thr;

[0207] Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

[0208] Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

[0209] Xaa₁₁ is Ala or Ser;

[0210] Xaa₁₂ is Ala or Lys;

[0211] Xaa₁₃ is Ala or Gin;

[0212] Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

[0213] Xaa₁₅ is Ala or Glu;

[0214] Xaa₁₆ is Ala or Glu;

[0215] Xaa₁₇ is Ala or Glu;

[0216] Xaa₁₉ is Ala or Val;

[0217] Xaa₂₀ is Ala or Arg;

[0218] Xaa₂₁ is Ala or Leu;

[0219] Xaa₂₂ is Phe, Tyr or naphthylalanine;

[0220] Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0221] Xaa₂₄ is Ala, Glu or Asp;

[0222] Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

[0223] Xaa₂₆ is Ala or Leu;

[0224] Xaa₂₇ is Ala or Lys;

[0225] Xaa₂₈ is Ala or Asn;

[0226] Z₁ is —OH,

[0227] —NH₂,

[0228] Gly-Z₂,

[0229] Gly Gly-Z₂,

[0230] Gly Gly Xaa₃₁-Z₂,

[0231] Gly Gly Xaa₃₁ Ser-Z₂,

[0232] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0233] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0234] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0235] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0236] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,

[0237] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or

[0238] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;

[0239] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro, homoproline,3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine; and

[0240] Z₂ is —OH or —NH₂;

[0241] provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈,Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and providedalso that, if Xaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄and Xaa₉ is Ala.

[0242] Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycineand N-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compoundsof formula (II) include those described in application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, identified therein in Examples 1-89 (“Compounds 1-89,”respectively), as well as those corresponding compounds identifiedtherein in Examples 104 and 105.

[0243] Preferred such exendin analogs include those wherein Xaa₁ is His,Ala or Norval.

[0244] More preferably Xaa₁ is His or Ala. Most preferably Xaa₁ is His.

[0245] Preferred are those compounds of formula (IV) wherein Xaa₂ isGly.

[0246] Preferred are those compounds of formula (IV) wherein Xaa₃ isAla.

[0247] Preferred are those compounds of formula (IV) wherein Xaa₄ isAla.

[0248] Preferred are those compounds of formula (IV) wherein Xaa₉ isAla.

[0249] Preferred are those compounds of formula (IV) wherein Xaa₁₄ isLeu, pentylglycine or Met.

[0250] Preferred compounds of formula (IV) are those wherein Xaa₂₅ isTrp or Phe.

[0251] Preferred compounds of formula (IV) are those where Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

[0252] Preferred are compounds of formula (IV) wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline and N-alkylalanine.

[0253] Preferably Z₁ is —NH₂.

[0254] Preferably Z₂ is —NH₂.

[0255] According to one aspect, preferred are compounds of formula (IV)wherein Xaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Alaor Gly; Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycineor Met; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

[0256] According to an especially preferred aspect, especially preferredcompounds include those of formula (IV) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₈, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided alsothat, if Xaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ andXaa₉ is Ala. Especially preferred compounds of formula (IV) includethose described in application Serial No. PCT/US98/24273, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds” as having the aminoacid sequence of SEQ. ID. NOS. 5-93 therein.

[0257] According to an especially preferred aspect, provided arecompounds of formula (IV) where Xaa₁₄ is Ala, Leu, Ile, Val orpentylglycine, more preferably Leu or pentylglycine, and Xaa₂₅ is Ala,Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine.These compounds will be less susceptible to oxidative degration, both invitro and in vivo, as well as during synthesis of the compound.

[0258] Formula V

[0259] Also within the scope of the present invention are narrowergenera of compounds having peptides of various lengths, for examplegenera of compounds which do not include peptides having a length of 28,29 or 30 amino acid residues, respectively. Additionally, the presentinvention includes narrower genera of compounds described in PCTapplication Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” and having particular amino acidsequences, for example, compounds of the formula (V) [SEQ. ID. NO:27]:

[0260] Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;

[0261] wherein:

[0262] Xaa₁ is His or Arg;

[0263] Xaa₂ is Gly or Ala;

[0264] Xaa₃ is Asp or Glu;

[0265] Xaa₅ is Ala or Thr;

[0266] Xaa₆ is Ala, Phe or naphthylalanine;

[0267] Xaa₇ is Thr or Ser;

[0268] Xaa₈ is Ala, Ser or Thr;

[0269] Xaa₉ is Asp or Glu;

[0270] Xaa₁₀ is Ala, Leu or pentylglycine;

[0271] Xaa₁₁ is Ala or Ser;

[0272] Xaa₁₂ is Ala or Lys;

[0273] Xaa₁₃ is Ala or Gln;

[0274] Xaa₁₄ is Ala, Leu or pentylglycine;

[0275] Xaa₁₅ is Ala or Glu;

[0276] Xaa₁₆ is Ala or Glu;

[0277] Xaa₁₇ is Ala or Glu;

[0278] Xaa₁₉ is Ala or Val;

[0279] Xaa₂₀ is Ala or Arg;

[0280] Xaa₂₁ is Ala or Leu;

[0281] Xaa₂₂ is Phe or naphthylalanine;

[0282] Xaa₂₃ is Ile, Val or tert-butylglycine;

[0283] Xaa₂₄ is Ala, Glu or Asp;

[0284] Xaa₂₅ is Ala, Trp, or Phe;

[0285] Xaa₂₆ is Ala or Leu;

[0286] Xaa₂₇ is Ala or Lys;

[0287] Xaa₂₈ is Ala or Asn;

[0288] Z₁ is —OH,

[0289] —NH₂,

[0290] Gly-Z₂,

[0291] Gly Gly-Z₂,

[0292] Gly Gly Xaa₃₁-Z₂,

[0293] Gly Gly Xaa₃₁ Ser-Z₂,

[0294] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0295] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0296] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0297] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0298] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or

[0299] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

[0300] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from thegroup consisting of Pro, homoproline, thioproline and N-methylylalanine;and

[0301] Z₂ is —OH or —NH₂;

[0302] provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; andpharmaceutically acceptable salts thereof

[0303] Formula VI

[0304] Additionally, the present invention includes narrower genera ofpeptide compounds described in PCT Application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” as having particular amino acid sequences, for example,compounds of the formula [VI] [SEQ. ID. NO:28]:

[0305] Xaa₁ Xaa₂ Xaa₃ Xaa₅ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;

[0306] wherein:

[0307] Xaa₁ is His or Ala;

[0308] Xaa₂ is Gly or Ala;

[0309] Xaa₃ is Ala, Asp or Glu;

[0310] Xaa₄ is Ala or Gly;

[0311] Xaa₅ is Ala or Thr;

[0312] Xaa₆ is Phe or naphthylalanine;

[0313] Xaa₇ is Thr or Ser;

[0314] Xaa₈ is Ala, Ser or Thr;

[0315] Xaa₉ is Ala, Asp or Glu;

[0316] Xaa₁₀ is Ala, Leu or pentylglycine;

[0317] Xaa₁₁ is Ala or Ser;

[0318] Xaa₁₂ is Ala or Lys;

[0319] Xaa₁₃ is Ala or Gln;

[0320] Xaa₁₄ is Ala, Leu, Met or pentylglycine;

[0321] Xaa₁₅ is Ala or Glu;

[0322] Xaa₁₆ is Ala or Glu;

[0323] Xaa₁₇ is Ala or Glu;

[0324] Xaa₁₉ is Ala or Val;

[0325] Xaa₂₀ is Ala or Arg;

[0326] Xaa₂₁ is Ala or Leu;

[0327] Xaa₂₂ is Phe or naphthylalanine;

[0328] Xaa₂₃ is Ile, Val or tert-butylglycine;

[0329] Xaa₂₄ is Ala, Glu or Asp;

[0330] Xaa₂₅ is Ala, Trp or Phe;

[0331] Xaa₂₆ is Ala or Leu;

[0332] Xaa₂₇ is Ala or Lys;

[0333] Xaa₂₈ is Ala or Asn;

[0334] Z₁ is —OH,

[0335] —NH₂,

[0336] Gly-Z₂,

[0337] Gly Gly-Z₂

[0338] Gly Gly Xaa₃₁-Z₂,

[0339] Gly Gly Xaa₃₁ Ser-Z₂,

[0340] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0341] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0342] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0343] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0344] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂

[0345] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂

[0346] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Ser-Z₂;

[0347] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro, homoproline,thioproline, or N-methylylalanine; and

[0348] Z₂ is —OH or —NH₂;

[0349] provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₈ are Ala; and provided that, if Xaa₁ is His,Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ is Ala; andpharmaceutically acceptable salts thereof.

[0350] Preferred compounds of formula (VI) include those wherein Xaa₁ isHis, Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

[0351] Preferred compounds of formula (VI) include those wherein Xaa₂ isGly.

[0352] Preferred compounds of formula (VI) include those wherein Xaa₄ isAla.

[0353] Preferred compounds of formula (VI) include those wherein Xaa₉ isAla.

[0354] Preferred compounds of formula (VI) include those wherein Xaa₄ isLeu, pentylglycine or Met.

[0355] Preferred compounds of formula (VI) include those wherein Xaa₂₅is Trp or Phe.

[0356] Preferred compounds of formula (VI) include those wherein Xaa₆ isAla, Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃is Ile or Val.

[0357] Preferred compounds of formula (VI) include those wherein Z₁ is—NH2.

[0358] Preferred compounds of formula (VI) include those wherein Xaa₃ ₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine.

[0359] Preferred compounds of formula (VI) include those wherein Xaa₃₉is Ser or Tyr, preferably Ser.

[0360] Preferred compounds of formula (VI) include those wherein Z2 is—NH2.

[0361] Preferred compounds of formula (VI) include those 42 wherein Z1is —NH2.

[0362] Preferred compounds of formula (VI) include those wherein Xaa21is Lys-NH2-R where R is Lys, Arg, C1-C10 straight chain or branchedalkanoyl.

[0363] Preferred compounds of formula (VI) include those wherein X1 isLys Asn, Lys-NHε-R Asn, or Lys-NHε-R Ala where R is Lys, Arg, C1-C10straight chain or branched alkanoyl. Preferred compounds of formula (VI)include those having an amino acid sequence described in PCT applicationSerial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” as being selected from SEQ. ID. NOS. 95-110 therein.

[0364] Formula VII

[0365] Also provided are compounds described in PCT applicationPCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VII) [SEQ. ID. NO. 29]:

[0366] Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ X₁-Z₁;

[0367] wherein

[0368] Xaa₁ is His, Arg or Tyr or 4-imidazopropionyl;

[0369] Xaa₂ is Ser, Gly, Ala or Thr;

[0370] Xaa₃ is Asp or Glu;

[0371] Xaa₅ is Ala or Thr;

[0372] Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

[0373] Xaa₇ is Thr or Ser;

[0374] Xaa₈ is Ala, Ser or Thr;

[0375] Xaa₉ is Asp or Glu;

[0376] Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

[0377] Xaa₁₁ is Ala or Ser;

[0378] Xaa₁₂ is Ala or Lys;

[0379] Xaa₁₃ is Ala or Gln;

[0380] Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

[0381] Xaa₁₅ is Ala or Glu;

[0382] Xaa₁₆ is Ala or Glu;

[0383] Xaa₁₇ is Ala or Glu;

[0384] Xaa₁₉ is Ala or Val;

[0385] Xaa₂₀ is Ala or Arg;

[0386] Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl or cycloalkylalkanoyl;

[0387] Xaa₂₂ is Phe, Tyr or naphthylalanine;

[0388] Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0389] Xaa₂₄ is Ala, Glu or Asp;

[0390] Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

[0391] Xaa₂₆ is Ala or Leu;

[0392] X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R,Lys-NH^(ε)—R Ala, Ala Lys-NH^(ε)—R where R is Lys, Arg, C¹-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl

[0393] Z₁ is —OH,

[0394] —NH₂,

[0395] Gly-Z₂,

[0396] Gly Gly-Z₂,

[0397] Gly Gly Xaa₃₁-Z₂,

[0398] Gly Gly Xaa₃₁ Ser-Z₂,

[0399] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0400] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0401] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0402] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0403] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or

[0404] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;

[0405] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from thegroup consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline,N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and

[0406] Z₂ is —OH or —NH₂;

[0407] provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀,Xaa₂₁, Xaa₂₄, Xaa₂₅, and Xaa₂₆ are Ala.

[0408] Also within the scope of the present invention arepharmaceutically acceptable salts of the compound of formula (VII) andpharmaceutical compositions including said compounds and salts thereof.

[0409] Preferred exendin analogs of formula (VII) include those whereinXaa₁ is His, Tyr or 4-imidazopropionyl. More preferably Xaa₁ is His.

[0410] Preferred are those compounds of formula (VII) wherein Xaa₁ is4-imidazopropionyl.

[0411] Preferred are those compounds of formula (VII) wherein Xaa₂ isGly.

[0412] Preferred compounds of formula (VII) are those wherein Xaa₁₄ isLeu, pentylglycine or Met.

[0413] Preferred compounds of formula (VII) are those wherein Xaa₂₅ isTrp or Phe.

[0414] According to one aspect, preferred are compounds of formula (VII)wherein Xaa₆ is Phe or naphthylalanine; and Xaa₂₂ is Phe ornaphthylalanine; and Xaa₂₃ is Ile or Val. More preferably, Z₁ is —NH₂.According to one aspect, especially preferred are such compounds offormula (VII) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independentlyselected from the group consisting of Pro, homoproline, thioproline andN-alkylalanine. More preferds, Z₂ is —NH₂. Preferred compounds offormula (VII) include those wherein X₁ is Lys Asn, Lys-NH^(ε)—R Asn, orLys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl. Preferred compounds of formula (VII) include compoundsdescribed in PCT application Serial No. PCT/US98/24210, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds” and identified thereinas Compound Nos. 62-69.

[0415] Preferred such exendin analogs include those wherein Xaa₁ is His,Ala or Norval. More preferably Xaa₁ is His or Ala. Most preferably Xaa₁is His.

[0416] Preferred are those compounds of formula (VII) wherein Xaa₂ isGly.

[0417] Preferred are those compounds of formula (VII) wherein Xaa₃ isAla.

[0418] Preferred are those compounds of formula (VII) wherein Xaa₄ isAla.

[0419] Preferred are those compounds of formula (VII) wherein Xaa₉ isAla.

[0420] Preferred are those compounds of formula (VII) wherein Xaa₁₄ isLeu, pentylglycine or Met.

[0421] Preferred compounds of formula (VII) are those wherein Xaa₂₅ isTrp or Phe.

[0422] Preferred compounds of formula (VII) are those where Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

[0423] Preferred are compounds of formula (VII) wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline and N-alkylalanine.

[0424] Preferably Z₁ is —NH₂.

[0425] Preferably Z₂ is —NH₂.

[0426] According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Alaor Gly; Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycineor Met; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

[0427] According to an especially preferred aspect, especially preferredcompounds include those of formula (VII) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (VII) include thosedescribed in PCT application Serial No. PCT/US98/24210, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds” and having the aminoacid sequences identified therein as SEQ. ID. NOS. 5-93.

[0428] According to an especially preferred aspect, provided arecompounds of formula (VII) where Xaa₁₄ is Ala, Leu, Ile, Val orpentylglycine, more preferably Leu or pentylglycine, and Xaa25 is Ala,Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine.These compounds will be less susceptible to oxidative degration, both invitro and in vivo, as well as during synthesis of the compound.

[0429] Formula VIII

[0430] Also provided are peptide compounds described in PCT ApplicationSerial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds”, including compounds of the formula (VIII) [SEQ. ID.NO:30]:

[0431] Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ X¹-Z₁;

[0432] wherein

[0433] Xaa₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or4-imidazopropionyl;

[0434] Xaa₂ is Ser, Gly, Ala or Thr;

[0435] Xaa₃ is Ala, Asp or Glu;

[0436] Xaa₄ is Ala, Norval, Val, Norleu or Gly;

[0437] Xaa₅ is Ala or Thr;

[0438] Xaa₆ is Phe, Tyr or naphthylalanine;

[0439] Xaa₇ is Thr or Ser;

[0440] Xaa₈ is Ala, Ser or Thr;

[0441] Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;

[0442] Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

[0443] Xaa₁₁ is Ala or Ser;

[0444] Xaa₁₂ is Ala or Lys;

[0445] Xaa₁₃ is Ala or Gln;

[0446] Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

[0447] Xaa₁₅ is Ala or Glu;

[0448] Xaa₁₆ is Ala or Glu;

[0449] Xaa₁₇ is Ala or Glu;

[0450] Xaa₁₉ is Ala or Val;

[0451] Xaa₂₀ is Ala or Arg;

[0452] Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C¹⁻¹⁰straight chain or branched alkanoyl or cycloalleyl-alkanoyl;

[0453] Xaa₂₂ is Phe, Tyr or naphthylalanine;

[0454] Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0455] Xaa₂₄ is Ala, Glu or Asp;

[0456] Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

[0457] Xaa₂₆ is Ala or Leu;

[0458] X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R,Lys-NH^(ε)—R Ala, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl

[0459] Z₁ is —OH,

[0460] —NH₂,

[0461] Gly-Z₂,

[0462] Gly Gly-Z₂,

[0463] Gly Gly Xaa₃₁-Z₂,

[0464] Gly Gly Xaa₃₁ Ser-Z₂,

[0465] Gly Gly Xaa₃₁ Ser Ser-Z₂,

[0466] Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,

[0467] Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,

[0468] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,

[0469] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,

[0470] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or

[0471] Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;

[0472] Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from thegroup consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline,N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and

[0473] Z₂ is —OH or —NH₂;

[0474] provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈,Xaa₉, Xaa₁₀, Xaa₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, are Ala; and provided also that, ifXaa₁ is His, Arg, Tyr, or 4-imidazopropionyl then at least one of Xaa₃,Xaa₄ and Xaa₉ is Ala.

[0475] Preferred compounds of formula (VIII) include those wherein Xaa₁is His, Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

[0476] Preferred compounds of formula (VIII) include those wherein Xaa₂is Gly.

[0477] Preferred compounds of formula (VIII) include those wherein Xaa₄is Ala.

[0478] Preferred compounds of formula (VIII) include those wherein Xaa₉is Ala.

[0479] Preferred compounds of formula (VIII) include those wherein Xaa₁₄is Leu, pentylglycine or Met.

[0480] Preferred compounds of formula (VIII) include those wherein Xaa₂₅is Trp or Phe.

[0481] Preferred compounds of formula (VIII) include those wherein Xaa₆is Ala, Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; andXaa₂₃ is Ile or Val.

[0482] Preferred compounds of formula (VIII) include those wherein Z₁ is—NH₂.

[0483] Preferred compounds of formula (VIII) include those whereinXaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine.

[0484] Preferred compounds of formula (VIII) include those wherein Xaa₃₉is Ser or Tyr, preferably Ser.

[0485] Preferred compounds of formula (VIII) include those wherein Z₂ is—NH₂.

[0486] Preferred compounds of formula (VIII) include those 42 wherein Z₁is —NH₂.

[0487] Preferred compounds of formula (VIII) include those wherein Xaa₂₁is Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

[0488] Preferred compounds of formula (VIII) include those wherein X₁ isLys Asn, Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg,C₁-C₁₀ straight chain or branched alkanoyl.

[0489] Preferred compounds of formula (VIII) include those described inPCT Application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” as having an amino acid sequenceselected from those identified therein as SEQ. ID. NOS. 95-110 .

[0490] Formula IX

[0491] Compounds particularly useful according to the present inventionare exendin analogs with agonist activity described in U.S. patentapplication Ser. No. 09/003,869, filed Jan. 7, 1998, entitled “Use ofExendins And Agonists Thereof For The Reduction of Food Intake”,including compounds of the formula (IX) [SEQ. ID. NO:31]:

[0492] Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Ser Lys Gln Xaa₉Glu Glu Glu Ala Val Arg Leu Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu Lys Asn Gly GlyXaa₁₄ Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Z

[0493] wherein:

[0494] Xaa₁ is His, Arg or Tyr;

[0495] Xaa₂ is Ser, Gly, Ala or Thr;

[0496] Xaa₃ is Asp or Glu;

[0497] Xaa₄ is Phe, Tyr or naphthalanine;

[0498] Xaa₅ is Thr or Ser;

[0499] Xaa₆ is Ser or Thr;

[0500] Xaa₇ is Asp or Glu;

[0501] Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

[0502] Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

[0503] Xaa₁₀ is Phe, Tyr or naphthalanine;

[0504] Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0505] Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine;

[0506] Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline,3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

[0507] Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

[0508] with the proviso that the compound does not have the formula ofeither SEQ. ID. NOS:12 or 14. Preferred N-alkyl groups forN-alkylglycine, N-alkylpentylglycine and N-alkylalanine include loweralkyl groups preferably of 1 to about 6 carbon atoms, more preferably of1 to 4 carbon atoms. Also useful in the present invention arepharmaceutically acceptable salts of the compounds of formula (IX).

[0509] Preferred exendin analogs include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

[0510] Preferred are those compounds wherein Xaa₂ is Gly.

[0511] Preferred are those compounds wherein Xaa₉ is Leu, pentylglycineor Met.

[0512] Preferred compounds include those wherein Xaa₁₃ is Trp or Phe.

[0513] Also preferred are compounds where Xaa₄ is Phe or naphthalanine;Xaa₁₁ is Ile or Val and Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

[0514] According to an especially preferred aspect, Xaa₅, Xaa₁₆ andXaa₁₇ are the same amino acid reside.

[0515] Preferred are compounds wherein Xaa₁₈ is Ser or Tyr, morepreferably Ser.

[0516] Preferably Z is —NH₂.

[0517] According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₄ isPhe or naphthalanine; Xaa₉ is Leu, pentylglycine or Met; Xaa₁₀ is Phe ornaphthalanine; Xaa₁₁ is Ile or Val; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₁₈ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

[0518] According to an especially preferred aspect, especially preferredcompounds include those of formula (IX) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly; Xaa₃ is Asp or Glu; Xaa₄ is Phe or napthylalanine; Xaa₅ isThr or Ser; Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu orpentylglycine; Xaa₉ is Leu or pentylglycine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile, Val or t-butyltylglycine; Xaa₁₂ is Glu orAsp; Xaa₁₃ is Trp or Phe; Xaa₁₄, Xaa₁₅, Xaa₁₆, and Xaa₁₇ areindependently Pro, homoproline, thioproline, or N-methylalanine; Xaa₁₈is Ser or Tyr: and Z is —OH or —NH₂; with the proviso that the compounddoes not have the formula of either SEQ. ID. NOS. 7 or 9. Morepreferably Z is —NH₂.

[0519] According to an especially preferred aspect, provided arecompounds where Xaa₉ is Leu, Ile, Val or pentylglycine, more preferablyLeu or pentylglycine, and Xaa₁₃ is Phe, Tyr or naphthylalanine, morepreferably Phe or naphthylalanine. These compounds are believed toexhibit advantageous duration of action and to be less subject tooxidative degration, both in vitro and in vivo, as well as duringsynthesis of the compound.

[0520] Formula X

[0521] Also provided are compounds described in PCT Application SerialNo. PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (X) [SEQ. ID. NO:32]:

[0522] Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Ser Lys Gln Xaa₉Glu Glu Glu Ala Val Arg Leu Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu X₁ Gly Gly Xaa₁₄Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Z

[0523] wherein:

[0524] Xaa₁ is His, Arg, Tyr or 4-imidazopropionyl;

[0525] Xaa₂ is Ser, Gly, Ala or Thr;

[0526] Xaa₃ is Asp or Glu;

[0527] Xaa₄ is Phe, Tyr or naphthylalanine;

[0528] Xaa₅ is Thr or Ser;

[0529] Xaa₆ is Ser or Thr;

[0530] Xaa₇ is Asp or Glu;

[0531] Xaa₈ is Leu, Ile, Val, pentylglycine or Met;

[0532] Xaa₉ is Leu, Ile, pentylglycine, Val or Met;

[0533] Xaa₁₀ is Phe, Tyr or naphthylalanine;

[0534] Xaa₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

[0535] Xaa₁₂ is Glu or Asp;

[0536] Xaa₁₃ is Trp, Phe, Tyr, or naphthylalanine; X₁ is Lys Asn, AsnLys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl or cycloalkylalkanoyl;

[0537] Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro, homoproline,3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine orN-alkylalanine;

[0538] Xaa₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH₂;

[0539] with the proviso that the compound does not have the formula ofeither SEQ. ID. NOS. 7 or 9. Suitable compounds of formula (X) includecompounds described in PCT Application Serial No. PCT/US98/16387, filedAug. 6, 1998, entitled “Novel Exendin Agonist Compounds” having theamino acid sequences of SEQ. ID. NOS. 37-40 therein.

[0540] Preferred exendin analogs of formula (X) include those whereinXaa₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa₁ is His or4-imidazopropionyl.

[0541] Preferred are those compounds of formula (X) wherein Xaa₂ is Gly.Preferred are those compounds of formula (X) wherein Xaa₉ is Leu,pentylglycine or Met.

[0542] Preferred are those compounds of formula (X) wherein Xaa₁₃ is Trpor Phe.

[0543] Preferred are those compounds of formula (X) wherein

[0544] X₁ is Lys Asn, or Lys-NH^(ε)—R Asn, where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl.

[0545] Also preferred are compounds of formula (X) wherein Xaa₄ is Pheor naphthylalanine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile or Valand Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine. According to an especiallypreferred aspect, Xaa₁₈ is Ser or Tyr. Preferred are those suchcompounds wherein Xaa₁₈ is Ser. Preferably, Z is —NH₂.

[0546] According to one preferred aspect, preferred are compounds offormula (X) wherein Xaa₄ is Phe or naphthylalanine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile or Val, X₁ is Lys Asn, or Lys-NH^(ε)—RAsn, where R is Lys, Arg, C₁-C₁₀ straight chain or branched alkanoyl andXaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine.

[0547] Exendins and exendin agonists that are peptides, such as exendinanalogs, described herein may be prepared through peptide purificationas described in, for example, Eng, et al., J. Biol. Chem. 265:20259-62,1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992, herebyincorporated by reference herein. Alternatively, exendins and exendinagonists that are peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al., J.Biol. Chem. 267:21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer aspreviously described and is well known in the art.

[0548] Exendins and exendin agonists that are peptides may also beprepared using recombinant DNA techniques, using methods now known inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d Ed., Cold Spring Harbor (1989). Alternatively, such compoundsmay be prepared by homogeneous phase peptide synthesis methods.Non-peptide compounds useful in the present invention may be prepared byart-known methods. For example, phosphate-containing amino acids andpeptides containing such amino acids, may be prepared using methodsknown in the art. See, e.g., Bartlett and Landen, Biorg. Chem.14:356-377 (1986). Methods for making and/or purifying GLP-1 and itsagonists, analogs, derivatives, variants, and fragments, as discussedpreviously, can also be utilized to make and/or purify exendins, theiragonists, analogs, derivatives, variants, and fragments thereof.

[0549] The compositions of the present invention may be used incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. The compositions ofthis invention can be administered in any effective, pharmaceuticallyacceptable form for warm blooded animals, including human and otheranimal subjects, e.g., in topical, lavage, oral, suppository,parenteral, or infusible dosage forms, as a topical, buccal, sublingual,pulmonary, or nasal spray or in any other manner effective to deliverthe agents. The route of administration will preferably be designed tooptimize delivery and/or localization of the agents.

[0550] In addition to the active compositions of the invention, thepharmaceutical composition may contain suitable excipients andauxiliaries that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Oral dosage formsencompass tablets, capsules, granules, solutions, and suspensions.Preparations that can be administered rectally include suppositories.Other dosage forms include suitable solutions for administrationparenterally or orally, and compositions which can be administeredbuccally or sublingually.

[0551] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself well known in the art. Forexample the pharmaceutical preparations may be made by means ofconventional mixing, granulating, dissolving, and lyophilizingprocesses. The processes to be used will depend ultimately on thephysical properties of the active ingredient used.

[0552] Suitable formulations for parenteral administration includeaqueous solutions of active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, including for example, sodiumcarboxymethyl cellulose, sorbitol and/or dextran. Such compositions mayalso comprise adjuvants such as preserving, wetting, emulsifying, anddispensing agents. They may also be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents into the compositions. They can also be manufacturedin the form of sterile solid compositions that can be dissolved orsuspended in sterile water, saline, or other injectable medium prior toadministration.

[0553] In addition to administration with conventional carriers, activeingredients may be administered by a variety of specialized deliverydrug techniques that are known to those of skill in the art, such asportable infusion pumps.

[0554] Additional formulations for administration may be made inaccordance with methods and amounts known in the art as set forth inRemington's Pharmaceutical Sciences, 18th Ed., Wiley Publishing (1990),the disclosure of which is herein incorporated by references in itsentirety.

[0555] The compositions of the present invention can be administeredalong with a pharmaceutically acceptable carrier in an amount sufficientto prevent arrhythmias and/or treat an active arrhythmia. The compoundsof this invention have extremely low toxicity and a low degree of sideeffects even at high doses. The dosing range of the compounds of thisinvention will vary depending on a number of factors, such as whether itis used for prophylaxis or treatment of arrhythmia, route ofadministration, desired dosing schedule, the physical health of thepatient, etc.

[0556] Although not limited to the following ranges and provided only asan illustration, exemplary dose ranges for use in the invention caninclude 0.001 pmol/kg to 500 nmol/kg per day depending on thecomposition selected. A lower limit of a dosage range can be about 0.001pmol/kg, 0.01 pmol/kg, 0.1 pmol/kg, 1 pmol/kg, 10 pmol/kg, or 100pmol/kg. An upper dosage range can be about 10 pmol/kg, 100 pmol/kg, 1nmol/kg, 10 nmol/kg, 100 nmol/kg, 250 nmol/kg or 500 nmol/kg. Thedesired dose will vary depending on the selected active composition. Thedesired dose will also depend upon other factors including the route ofadministration and the formulation. For example, continuous infusion aswell as bolus doses and sustained release formulations are contemplated.Routes of administration include intramuscular, intravenous,subcutaneous, intradermal, transdermal, intraarticular, intrathecal andthe like. Mucosal delivery is also contemplated. These routes include,but are not limited to, oral, nasal, sublingual, rectal, pulmonary andbuccal routes, which may include administration of the peptide inliquid, semi-solid or solid form.

[0557] Exemplary doses for continuous infusion by intravenous (I.V.) canbe about 0.1 pmol/kg/min to 10 pmol/kg/min and by subcutaneous (s.c.)about 0.1 pmol/kg/min to 75 pmol/kg/min., and for single injection(bolus) by I.V. about 0.1 nmol/kg to 2.0 nmol/kg and s.c. about 0.1nmol/kg to 100 nmol/kg. The foregoing doses may be administered as asingle dose or may be divided into multiple doses for administration.The peptides of this invention may be administered once to several timesdaily.

[0558] While a preferred method of administration of a GLP-1 peptide maybe through a continuous application, other forms of delivery asdescribed above are also contemplated. However, an exemplary dosing ratecan be within a range of from about 1 to about 10 pmol/kg per minute ofGLP-1 delivered by sustained release subcutaneous, intramuscular,interperitoneal, injected depot with sustained release, deep lunginsufflation, as well as by intravenous, buccal, patch or othersustained release delivery methods. Degradation-resistant GLP-1 analogs,derivatives or variants, exendins, analogs, derivatives or variants, andother molecules of the invention need not be delivered continuously, butare suitable for bolus or sustained release dosing and may be at dosesmuch lower than those described.

[0559] Other drugs besides compositions of the invention which arecompatible with the carrier ingredients may also be incorporated intothe pharmaceutical formulations. Such drugs may be readily ascertainedby those of ordinary skill in the art and may include, for instance,anti-inflammatory agents, diuretics, vasodilators, etc.

[0560] It is understood that the present invention contemplates the useof not only the above-stated active forms of the compositions of theinvention, but also includes the prodrugs (proforms) which metabolize tothe compound and biologically active salt forms thereof, as well asoptical isomers which provide the same pharmaceutical results.

[0561] The compositions of the invention may also be used in combinationwith agents known in the art that enhance the half-life in vivo ofpeptide in order to enhance or prolong the biological activity of thepeptide. For example, a molecule or chemical moiety may be covalentlylinked to the composition of the present invention before administrationthereof. Alternatively, the enhancing agent may be administeredconcurrently with the composition. Still further, the agent may comprisea molecule that is known to inhibit the enzymatic degradation of thecompositions of the invention that may be administered concurrently withor after administration of the composition. Such a molecule may beadministered, for example, orally, by injection, or any other meansknown in the art.

[0562] In accordance with this invention, compositions of the inventionin combination with a pharmaceutically acceptable carrier are preferablyadministered within the first four hours following an ischemic event inorder to prevent the occurrence of cardiac arrhythmia. Compositions ofthe invention can be co-administered with glucose (5%) if required tomaintain blood glucose levels ≧5 mM (to maintain efficient insulinsecretion). Similarly, co-administration of potassium (K⁺) may beconsidered, depending on the extent to which activation of the membraneNa⁺/K⁺ ATPase leads to a shift of K⁺ into the intracellular space.

[0563] With respect to reperfusion, treatment with compositions of theinvention should be commenced concurrently or as soon as possiblefollowing therapies that reestablish flow in an artery that wasobstructed by a blood clot (e.g., thromolytic therapy) or otherobstructive materials, or following an intervention, such asangioplasty, coronary bypass grafting, or placement of an intracoronarystent. Therapy should continue thereafter. In the case of cardiacsurgery, the treatment should preferably commence 12-24 hours prior tosurgery, during surgery from the onset of anesthesia until aorticcrossclamping, and immediately after unclamping for a period of at least72 hours postoperatively. As earlier explained, co-administration of afree radical scavenger or antioxidants will further aid reperfusionrecovery.

1 49 1 30 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser TyrLeu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val LysGly Arg 20 25 30 2 30 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 2 Ala Glu Gly Thr Phe Thr Ser Asp Val Ser SerTyr Leu Glu Gly Gln 1 5 10 15 Ala Ala Xaa Glu Phe Ile Ala Trp Leu ValLys Gly Arg Xaa 20 25 30 3 30 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Ser Tyr Leu Glu Gly Gln 1 5 10 15 Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Xaa Gly Arg Xaa 20 25 30 4 4 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 4 Ser Asp Val Ser 15 5 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 5 Thr Ser Asp Val Ser 1 5 6 6 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 6 Phe Thr Ser AspVal Ser 1 5 7 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 7 Thr Phe Thr Ser Asp Val Ser 1 5 8 8 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide8 Gly Thr Phe Thr Ser Asp Val Ser 1 5 9 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 9 Glu Gly Thr PheThr Ser Asp Val Ser 1 5 10 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 10 Ala Glu Gly Thr Phe Thr Ser AspVal Ser 1 5 10 11 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 11 His Ala Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala TrpLeu Val Lys Xaa Xaa Xaa 20 25 30 12 39 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 12 His Ser Asp GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaPro Pro Pro Ser 35 13 31 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 13 Asp Leu Ser Lys Gln Met Glu GluGlu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly ProSer Ser Gly Ala Pro Pro Pro Ser 20 25 30 14 39 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 14 His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaPro Pro Pro Ser 35 15 38 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 15 His Ser Asp Ala Thr Phe Thr AlaGlu Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln Lys Tyr LeuGlu Ser Ile Leu Gly Ser Ser Thr Ser 20 25 30 Pro Arg Pro Pro Ser Ser 3516 37 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 16 His Ser Asp Ala Thr Phe Thr Ala Glu Tyr Ser Lys LeuLeu Ala Lys 1 5 10 15 Leu Ala Leu Gln Lys Tyr Leu Glu Ser Ile Leu GlySer Ser Thr Ser 20 25 30 Pro Arg Pro Pro Ser 35 17 35 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 17 His SerAsp Ala Ile Phe Thr Glu Glu Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 LeuAla Leu Gln Lys Tyr Leu Ala Ser Ile Leu Gly Ser Arg Thr Ser 20 25 30 ProPro Pro 35 18 35 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 18 His Ser Asp Ala Ile Phe Thr Gln Gln TyrSer Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln Lys Tyr Leu Ala SerIle Leu Gly Ser Arg Thr Ser 20 25 30 Pro Pro Pro 35 19 30 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 19 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 25 30 20 30 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide20 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 25 3021 28 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 21 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnMet Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn 2025 22 39 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 22 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnLeu Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys AsnGly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 23 28 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide23 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20 25 24 28 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide24 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu 1 510 15 Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn 20 25 25 38 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide25 Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 510 15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 2025 30 Xaa Xaa Xaa Xaa Xaa Xaa 35 26 39 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 26 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa XaaXaa Xaa Xaa Xaa 35 27 38 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 27 Xaa Xaa Xaa Gly Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa 3528 39 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 28 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 29 40 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide29 Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 510 15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 2025 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 30 41 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaAla Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 31 39 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 31 Xaa Xaa Xaa GlyThr Xaa Xaa Xaa Xaa Xaa Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala ValArg Leu Xaa Xaa Xaa Xaa Leu Lys Asn Gly Gly Xaa Ser 20 25 30 Ser Gly AlaXaa Xaa Xaa Xaa 35 32 40 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 32 Xaa Xaa Xaa Gly Thr Xaa Xaa XaaXaa Xaa Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Xaa XaaXaa Xaa Leu Xaa Xaa Xaa Gly Gly Xaa 20 25 30 Ser Ser Gly Ala Xaa Xaa XaaXaa 35 40 33 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 33 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu Xaa Gly 1 5 10 15 Gln Ala Ala Lys Xaa Phe Ile Ala TrpLeu Val Lys Gly Arg Xaa 20 25 30 34 31 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 34 Xaa Xaa Glu GlyThr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly 1 5 10 15 Gln Ala AlaLys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30 35 5 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide35 Gly Gly Xaa Ser Ser 1 5 36 6 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 36 Gly Gly Xaa Ser Ser Gly 1 5 377 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 37 Gly Gly Xaa Ser Ser Gly Ala 1 5 38 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 38 Gly Gly Xaa SerSer Gly Ala Xaa 1 5 39 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 39 Gly Gly Xaa Ser Ser Gly Ala XaaXaa 1 5 40 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 40 Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 4111 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 41 Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Xaa 1 5 10 42 5 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide42 Gly Gly Xaa Ser Ser 1 5 43 6 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 43 Gly Gly Xaa Ser Ser Gly 1 5 447 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 44 Gly Gly Xaa Ser Ser Gly Ala 1 5 45 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 45 Gly Gly Xaa SerSer Gly Ala Xaa 1 5 46 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 46 Gly Gly Xaa Ser Ser Gly Ala XaaXaa 1 5 47 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 47 Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 4811 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 48 Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Ser 1 5 10 49 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide49 Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Xaa 1 5 10

What is claimed is:
 1. A method for preventing and treating arrhythmiascomprising: administering to an individual in need of such treatment aneffective amount of a composition which includes a compound which bindsto a receptor for glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of aforementionedcompounds, and biologically active fragments thereof.
 2. The method ofclaim 1 wherein the composition comprises a GLP-1, an agonist, ananalog, a derivative, a variant, or a biologically active fragmentthereof.
 3. The method of claim 1 wherein the composition comprises anexendin, an agonist, an analog, a derivative, a variant, or abiologically active fragment thereof.
 4. The method of claim 1 whereinthe composition is administered in a dose of from about 0.1 pmol/kg/min.up to about 10 pmol/kg/min.
 5. The method of claim 1 wherein thecomposition is administered in a dose of from about 0.01 pmol/kg to 20nmol/kg.
 6. The method of claim 1 wherein the composition isadministered as a single injection in a dose of from about 0.005 nmol/kgto 20 nmol/kg.
 7. The method of claim 1 wherein the composition isadministered concurrently with glucose.
 8. The method of claim 1 whereinthe composition is administered concurrently with potassium.
 9. Themethod of claim 1 wherein the composition is administered concurrentlywith a free radical scavenger.
 10. The method of claim 1 wherein thecomposition is administered within four hours of an ischemic event. 11.The method of claim 10 wherein the composition continues to beadministered following the ischemic event.
 12. The method of claim 1wherein the composition is administered concurrently or as soon aspossible following therapies that reestablish flow in an artery that hasbeen obstructed.
 13. The method of claim 1 wherein the composition isadministered following a cardiac intervention selected from the groupconsisting of angioplasty, coronary bypass grafting, and placement of anintracoronary stent.
 14. The method of claim 13 wherein the compositioncontinues to be administered following the intervention.
 15. The methodof claim 1 wherein the composition is administered to treat ventriculararrhythmias.
 16. The method of claim 15 wherein the ventriculararrhythmia is caused by a condition selected from the group consistingof cardiac ischemia, cardiac ischemia-reperfusion, and congestive heartfailure.
 17. A method of metabolic intervention with a composition thatincludes a compound which binds to a receptor for glucagon-likepeptide-1, an incretin, a glucagon-like peptide-1 (GLP-1), an exendin,or an agonist, an analog (preferably an agonist analog), a derivative,or a variant of any of aforementioned compounds, and fragments thereofto prevent or treat cardiac arrhythmias, said method comprising:administering to an individual in need of such treatment an effectiveamount of a composition which includes a compound which binds to areceptor for glucagon-like peptide-1, an incretin, a glucagon-likepeptide-1 (GLP-1), an exendin, or an agonist, an analog (preferably anagonist analog), a derivative, or a variant of any of aforementionedcompounds, and biologically active fragments thereof.